Cerebral infarction remains a significant cause of morbidity and mortality, with limited treatment options available. New therapies under development, while promising, are frequently only useful when administered immediately after the onset of cerebral ischemia. The pathophysiology of cerebral ischemia is poorly understood, but it is clear that a component of the neuronal death that occurs after an ischemic insult is delayed for hours, and even days. New therapies need to be developed that will interrupt the delayed components of neuronal death after ischemia. Such therapies could be of real clinical benefit if they are effective when administered hours after ischemia. The heat stress response is a highly conserved response to stress seen in organisms from bacteria to man. Recent data shows that animals, and cultured cells, that undergo a heat stress before an ischemic event have significantly less cell death than do non heat-stressed animals. It is likely that the heat stress response affects the delayed component of neuronal death. Understanding the mechanisms of neuroprotection by heat stress could provide valuable clues to new therapies for stroke and, possibly, other neurologic diseases. The goal of the proposed project is to determine the relative contributions of neuronal and glial heat shock protein 70 (hsp70) expression in reducing neuronal death in an in vitro model of ischemic damage. Hsp70 has been shown to mediate the protective effects of heat stress against subsequent severe thermal insults, and has been suggested to mediate the protective effects of heat stress in in vivo models of ischemia. We have observed that heat stress protects cultured cortical neurons from a subsequent oxygen glucose deprivation injury. However, in most studies of brain hsp70 expression after hyperthermia, and in studies in cultured cells, hsp70 expression after heat stress is predominantly glial. We propose that glial hsp70 expression plays a role in protecting neurons from damage during oxygen glucose deprivation. We will test this hypothesis by correlating glial hsp70 expression with neuroprotection. Glial hsp70 expression will be manipulated by pharmacologic means and by varying the heat stress conditions and the interval between heat stress and oxygen glucose deprivation. We will determine if glial hsp70 expression is sufficient for neuroprotection by transfecting an hsp70 containing plasmid into glia; neurons will then be plated on these glia and their susceptibility to oxygen glucose deprivation injury determined. We will test the hypothesis that hsp70 synthesis is necessary for neuroprotection by heat stress by inhibiting hsp70 expression using quercetin (an inhibitor of hsp70 expression) and antisense oligonucleotides. Finally, we will test two potential mechanisms through which glial hsp70 expression could ameliorate neuronal damage in oxygen glucose deprivation. Glial glutamate uptake will be compared between control and heat stressed cultures and the ability of heat stress to protect neurons from oxidative stress will be determined. Understanding the mechanisms of heat stress induced neuroprotection may provide valuable clues into the pathophysiology of ischemia and may help us understand how neurons and glia interact in pathologic conditions.